Increasing Population Status of Broad-Snouted Caiman (Caiman latirostris) Based on Sustainable Use Strategies in a Managed Protected Area in Santa Fe, Argentina.

People and crocodilians have engaged in interactions since the earliest human settlements. After many years of escalating non-regulated use, coupled with emerging threats such as habitat modification, environmental pollution, and the exponential growth of human populations, natural crocodilian populations have been pushed to the brink of extinction. To prevent this, various initiatives have implemented strategies to prevent local extinction. Reinforcing wild populations through reintroductions and head-starting programs has been considered the safest approach to recovering crocodilian populations. Subsequently, the concept of sustainability emerged. In the case of many historically exploited crocodilian species, it became evident that rational utilization need not adversely affect population status; on the contrary, there were clear signs of recovery when local communities were involved. In 1990, the broad-snouted caiman (Caiman latirostris) was in a critical state due to decades of poaching and the aforementioned threats. In response, the "Monitoring and restocking program of the broad-snouted caiman for management purposes", commonly known as Proyecto Yacaré, was established to study population abundance and assess the biological, ecological, and social response to this management methodology. As a result of the eggs's harvest, a proportion equivalent to or greater than what would have survived in natural conditions was released into the original habitat where the eggs were collected. The surplus was allocated for leather and meat production with a focus on economic and environmentally sustainable practices, without affecting biodiversity or threats to the managed species. The in situ work carried out by this program has been crucial for the recovery of broad-snouted caiman populations under the "incentives for conservation" system, whereby local communities receive benefits for nest identification and egg harvesting. Over time, conservation incentives have become significant contributors to regional economies. This study illustrates how C. latirostris populations increased due to the implementation of egg harvesting by local communities in a natural reserve for management purposes. This population increase was detected through continuous night monitoring and an analysis of the number of nests in the area. Currently, C. latirostris populations have transitioned from being among the most threatened to becoming one of the most abundant throughout their distribution area. Based on the analyzed information, we may affirm that the commercial value of these species is one of the most relevant aspects contributing to the sustainability of these programs, primarily due to the change in perception generated among local communities. Therefore, identifying and controlling factors affecting these programs are fundamental for the conservation of these species.

A true facultative carnivore? Effects of replacing ground chicken carcasses with soybean meal on the growth of captive broad-snouted caiman (Caiman latirostris) and its economics implications.

A specific diet for broad-snouted caiman, Caiman latirostris has not been designed despite the value of farm-raised caiman as an aquaculture product. To fill this gap, the objectives of this study were to evaluate the performance dietary replacement of ground chicken carcasses by of soybean meal (SM) as diet complement for C. latirostris. We conducted a 3-month growth trial to determine effects of graded levels of dietary SM on caiman growth as measured by increase in body length, body weight gain, food consumption (FC) and food conversion rate (FCR). Forty-eight hatchling caimans were fed with diets, composed primarily of practical feed ingredients, with 0, 25, or 40% dietary SM. Diets were fed three times per week for 90 days under temperature controlled. Body lengths and body weights were measured at 30-day intervals, and FC samples were taken between the 31-60-day interval. The results of this study indicate that the inclusion of SM in the diet of C. latirostris at levels of 25% increases in body length, body weight gain, FC and improve the FCR indicated that a concentration of 25% dietary SM (as fed) was adequate for growth of caimans under the conditions of this study. Results suggest that SM have a real nutritional contribution in the diet of broad-snouted caiman and can be used as an ingredient of the diet of the crocodilians raised in captivity, reducing production costs for sustainable use and conservation programs of this species.

Blood biochemistry parameters of Broad snouted caiman, Caiman latirostris subjected to stress conditions, appliying micro-volume techniques.

Plasma biochemistry values represent a crucial and minimally invasive tool for evaluating nutritional and physiological condition of the animal. Biochemical data provides valuable information in those species exposed to environmental stressors. In this study, we aimed to apply for the first time micro-volume plasma tests to measured total plasma protein, albumin, and glucose in juvenile Caiman latirostris and to compare our results with other studies reporting crocodilian biochemistry values. We found that caimans from heat treatment were heavier, longer and had higher levels of glucose than those from the control group. Conversely, food restricted caimans had less growth, body condition and had 32.1% lower albumin concentrations than those from the control group. While no significant differences were observed in any parameters measured from water restricted caimans compared with control group. The values obtained for each of the biochemical determinations were lower than those previously reported for adult of C. latirostris, but similar finding for protein concentration compared with Alligator mississippiensis. Micro technique offers the advantage of increasing information and at the same time a greater physiological understanding of the crocodilians species.

Genotoxicity induced by widely used pesticide binary mixtures on Caiman latirostris (broad-snouted caiman).

In this study, we investigated the effects of three binary mixtures of pesticide formulations commonly used in soybean crop: Cypermethrin Atanor® (25% -CYP), Chlorpyrifos Lorsban 48E® (48% -CPF) and Glyphosate Roundup® Full II (66.2% -GLY) on broad-snouted caiman (Caiman latirostris) hatchlings exposed by voluntary immersion under controlled condition. Genotoxicity was evaluated in peripheral blood erythrocytes through the micronucleus (MN) test and other nuclear abnormalities (NAs) and besides, growth of caiman was analyzed in each experimental group. The results showed that pesticide formulations tested, at concentrations similar to those recommended for application in the field, induced an increase in the frequency of micronucleus (FMN; p = 0.001) and Notched nuclei (NN; p = 0.010) in the mixture CYP + CPF, while an increase in the frequency of buds and NN was observed in the mixture of GLY + CYP (pbuds = 0.016 and pNN = 0.021), compared to the vehicle control (VC). On the contrary, a possible antagonistic action was observed between the components in the mixture GLY + CPF. Growth was not affected in any exposed groups (p > 0.05). There was a clutch effect in the frequency of binucleated erythrocytes (BiN; p = 0.011), total length (TL; p = 0.001) and snout-vent length (SVL; p = 0.031). Biomarkers used in this study are considered important predictive tools for the evaluation of xenobiotics. In this study, we demonstrated genotoxicity of pesticide mixtures under conditions that simulate the real situation of exposure suffered by caiman and other wild species in Argentina.

Effects of cypermethrin (pyrethroid), glyphosate and chlorpyrifos (organophosphorus) on the endocrine and immune system of Salvator merianae (Argentine tegu).

Several geographical areas where Salvator merianae is distributed in Argentina are included in regions with agricultural activity and exposed to pesticide formulations. Some pesticides could affect defense mechanisms being able alter structures of some components of immune and endocrine systems. To assess the potential effects of pesticides in this reptile under seminatural conditions, on the immune system and endocrine responses in S. merianae we analyzed several blood parameters. Total (TWBCC), differential (DWBCC) white blood cells count, heterophils/lymphocytes index (H/L), lobularity index (LI), natural antibodies (NAbs) titres, complement system (CS), and corticosterone concentration were analyzed in animals exposed to a mixture of cypermethrin (25%), glyphosate (66.2%) and chlorpyrifos (48%) formulations. In addition, body size was considered in these analyzes. TWBCC and NAbs revealed lower values in organisms exposed to pesticides respect to a control indicating a possible immunosuppression effect. Besides, the LI showed a greater number of lobes in organism exposed demonstrating symptoms of chronic infection. In addition, we observed a reduced growth in these animals possibly related to a less energy investment in body mass to maintain an active defense against pesticides. Finally, we found high levels of plasma corticosterone in animals exposed to mix formulation that could demonstrate neuroendocrine axis activation. Other parameters like DWBCC, H/L index and activity of CS showed no differences in treated animals respect to control group, which could indicate low sensibility of these parameters to the concentration of pesticides used. Our results provide evidence of the toxic effects of pesticides on different immune system parameters, but also a trade-off among these parameters, corticosterone levels and growth. In this way, we can conclude that the formulated pesticides applied widely and constantly in the areas occupied by S. merianae, would be affecting its immune and endocrine systems and therefore its ability to defend against external agents. This kind of studies is of great interest to know the possible responses of wild species to anthropogenic disturbances such as pesticide contamination.

Analysis of morphological variability and heritability in the head of the Argentine Black and White Tegu (Salvator merianae): undisturbed vs. disturbed environments.

The heterogeneity of biotic and abiotic factors influencing fitness produce selective pressures that promote local adaptation and divergence among different populations of the same species. In order for adaptations to be maintained through evolutionary time, heritable genetic variation controlling the expression of the morphological features under selection is necessary. Here we compare morphological shape variability and size of the cephalic region of Salvator merianae specimens from undisturbed environments to those of individuals from disturbed environments, and estimated heritability for shape and size using geometric morphometric and quantitative genetics tools. The results of these analyzes indicated that there are statistically significant differences in shape and size between populations from the two environments. Possibly, one of the main determinants of cephalic shape and size is adaptation to the characteristics of the environment and to the trophic niche. Individuals from disturbed environments have a cephalic region with less shape variation and also have a larger centroid size when compared to individuals from undisturbed environments. The high heritability values obtained for shape and size in dorsal view and right side view indicate that these phenotypic characters have a great capacity to respond to the selection pressures to which they are subjected. Data obtained here could be used as an important tool when establishing guidelines for plans for the sustainable use and conservation of S. merianae and other species living in disturbed areas.

Baseline values of immunologic parameters in the lizard Salvator merianae (Teiidae, Squamata).

The genus Salvator is widely distributed throughout South America. In Argentina, the species most abundant widely distributed is Salvator merianae. Particularly in Santa Fe province, the area occupied by populations of these lizards overlaps with areas where agriculture was extended. With the aim of established baseline values for four immunologic biomarkers widely used, 36 tegu lizards were evaluated tacking into account different age classes and both sexes. Total leukocyte counts were not different between age classes. Of the leucocytes count, eosinophils levels were higher in neonates compared with juvenile and adults; nevertheless, the heterophils group was the most prevalent leukocyte in the peripheral blood in all age classes. Lymphocytes, monocytes, heterophils, azurophils and basophils levels did not differ with age. Natural antibodies titres were higher in the adults compared with neonates and juveniles lizards. Lastly, complement system activity was low in neonates compared with juveniles and adults. Statistical analysis within each age group showed that gender was not a factor in the outcomes. Based on the results, we concluded that S. merianae demonstrated age (but not gender) related differences in the immune parameters analyzed. Having established baseline values for these four widely-used immunologic biomarkers, ongoing studies will seek to optimize the use of the S. merianae model in future research.

Mating System of Caiman yacare (Reptilia: Alligatoridae) Described from Microsatellite Genotypes.

The yacare caiman (Caiman yacare) is a reptile from South America and 1 of the 2 crocodilian species present in Argentina. The degradation of their natural habitat and strong hunting pressure led to a sharp numerical decline of wild populations. Nowadays, C. yacare is included in Appendix II of CITES, and ranching practices in some areas in Argentina are helping hatching success. In this context, it is important to better understand the population structure and mating system of the species. To do this, we amplified 10 microsatellite markers (SSRs) in 148 individuals of 13 C. yacare nests. All of the markers were polymorphic with 2­12 alleles per locus, with allelic sizes ranging between 154 and 400 bp and medium levels of polymorphism (Ho = 0.152­0.551 and He = 0.221­0.621). We were able to determine the maternal genotype in 9 out of 13 nests. In 6 of them we found more than 1 paternal genotype, with a maximum of 3 fathers for a single nest. This study is the first to provide evidence of multiple paternity behavior. These findings will be useful to improve management and conservation strategies for the species.

Total and differential white blood cell counts in Caiman latirostris after in ovo and in vivo exposure to insecticides.

Agricultural activities associated mainly with soybean crops affect the natural environment and wildlife by habitat destruction and the extensive use of agrochemicals. The aim of this study was to evaluate immunotoxic effects of the insecticides cypermethrin (CYP) and endosulfan (END) in Caiman latirostris analyzing total blood cell count (TWBC) and differential white blood cell count (DWBC) after in ovo and in vivo exposure. Eggs (in ovo) and hatchlings (in vivo) from nests harvested in natural habitats were artificially incubated and reared under controlled conditions in the Proyecto Yacaré (Gob.Santa Fe/MUPCN) facilities. Exposure of embryos was performed by topication on the eggshell during the first stage of development. The treatments were distilled water (negative control; NC), ethanol (vehicle control; VC), four groups treated with different concentrations of CYP and four groups with END. In vivo exposure was performed by immersion; treatments were NC, VC, two groups exposed to CYP and two to END. After embryonic exposure to the insecticides, no differences were found in TWBC or DWBC among the neonates exposed to pesticides versus controls. In the in vivo scenario, similar results were obtained for TWBC, but DWBC data showed differences between NC hatchlings and CYP-1 hosts for heterophil, lymphocyte and monocyte levels, and between NC and END-1 hosts for lymphocyte and monocyte levels. Research on the effects of pesticide exposure on this species is of special interest not only to assess the impact on caiman populations, but also to further characterize the species as a potential sentinel of ecosystem health.

Phenotypic variability and heritability of the cephalic region of Caiman latirostris.

The study of the cephalic shape of crocodilian is relevant in the fields of ecology, systematics, evolution, and conservation. Therefore, the integration of geometric analysis within quantitative genetics allows the evaluation of the inheritable shape components. In this study, the dorsal cephalic region of 210 Caiman latirostris hatchlings was analyzed from seven populations in Santa Fe, Argentina, to detect intra-, and inter-population phenotypic variability, and to determine the heritability of biological shape and size, using newly available geometric morphometric tools. The principal component analysis showed two configurations of cephalic shape that could be related to sexual dimorphism. In the canonical variate analysis, Procrustes distances between groups indicated that there are differences in shape among populations. Furthermore, the method of partial least squares indicated a covariation between cephalic shape and environmental variables. Regarding to CS of the skull we found significant differences among populations, moreover the partial least squares was also significant. Estimates of the heritability of shape and size were high, indicating that the components of these features are susceptible to the selection.

Effects of in vivo exposure to Roundup® on immune system of Caiman latirostris.

The present study aimed to evaluate the effect of Roundup(®) (RU, glyphosate-based formulation) on some parameters of the immune system and growth of Caiman latirostris. Seventy-two caimans (20-day-old) from Proyecto Yacaré (Gob. Santa Fe/MUPCN) were used. Two groups were exposed for 2 months to different concentrations of RU (11 or 21 mg/L; taking into account the concentration recommended for its application in the field), while one group was maintained as control. The RU concentration was progressively decreased through the exposure period to simulate glyphosate degradation in water. Animals were measured and weighed at the beginning and end of the experiment, and blood samples taken after exposure to determine total and differential white blood cell (WBC) counts as well as total protein concentration (TPC), and for performing protein electrophoresis. The results showed that, compared against control hosts, there was a decrease in WBC counts, a higher percentage of heterophils, a higher TPC (with a low percentage of F2 protein fraction), and a negative effect on growth in the young caimans exposed to RU. These results demonstrate that in vivo exposure to RU induced alterations in the selected immune parameters, plasma proteins, and growth of caimans, thereby providing relevant information about the effects of this type of pesticide in this important species in the Argentinian wetlands.

Baseline values of micronuclei and comet assay in the lizard Tupinambis merianae (Teiidae, Squamata).

The Micronucleus test (MN) and Comet assay (CA) are currently the most widely used methods that allow the characterization of DNA damage induced by physical and chemical agents in wild species. The continuous expansion of the cultivated areas in Argentina, since the introduction of transgenic crops, mainly soy, in association with the increased use of pesticides, transformed deeply the natural environments where the lizard Tupinambis merianae (tegu lizard) occurs. Despite the fact that reptiles have shown to be excellent bioindicators of environmental contaminants, there is no record of genotoxicity studies in T. merianae. The aim of the present study was to adjust the MN test and CA protocols to be applied in erythrocytes of T. merianae, and determine the baseline values of DNA damage in this species. We used 20 adult lizards (10 males: 10 females) from Estación Zoológica Experimental "Granja La Esmeralda" (Santa Fe, Argentina). Peripheral blood samples were collected from all animals and the MN test and CA applied according to the protocols established for other reptilian species. We test critical parameters of CA protocol (cell density, unwinding and electrophoresis times) using increasing concentrations of H2O2 (10, 25 and 50 µM) as a known genotoxic agent to induce DNA damage. Based on this, we determined the most suitable conditions for the CA in this species: a cell density of 4×10(3) erythrocytes per slide, 10 min of unwinding and 15 min of electrophoresis at 0.90 V/cm approximately. The baseline frequency of micronuclei (BFMN=MN/1000 erythrocytes counted) determined for this species was 0.95±0.27 and the basal damage index (BDI: calculated from 100 comet images classified in arbitrary units)=103.85±0.97. No differences were observed between sexes in the BFMN or BDI (p>0.05), and no relation was found between baseline values and length or weight of the analyzed animals (p>0.05). These results demonstrated the sensitivity of both biomarkers of genotoxicity to be applied in erythrocytes of this species, with baseline values comparable to those reported in other reptilian species. These results allow us to propose the tegu lizard for future in vivo studies to assess the genotoxicity of different agents, including those possibly affecting it in its natural geographic distribution.

Ultraviolet radiation on innate immunity and growth of broad-snouted caiman (Caiman latirostris): implications for facilities design.

Sunlight is a key environmental factor in almost all ecosystems, and it is necessary for many physiological functions. Many vertebrates require ultraviolet (UV) radiation to perform different physiological processes. Artificial light is used to supplement UV in captive animals, through appropriate photoperiods and UV wavelengths. Previous studies reported that repeated exposure to artificial UV radiation may cause damage to the immune system. Taking into account the importance of UV effects and the serum complement system, the relationship between them was investigated. The study lasted 90 days and was carried out in plastic chambers. Ninety six broad-snouted caiman (C. latirostris) were assigned to four treatment groups with two replicates each: total darkness (TD), 8 hr per day (8 hr) and 16 hr per day (16 hr) of artificial UV/visible light exposure, and normal photoperiod of natural light (NP). Snout-vent length was measured to determine animal growth. Hemolytic assays were performed to evaluate the effects of artificial UV/visible light, TD, and NP on the serum complement system. Results showed that animals grew more in the NP group. The capacity of C. latirostris serum to hemolyze sheep red blood cells was higher in the NP group than when they are maintained in constant light-dark cycles (8 and 16 hr) or in TD. These data demonstrate that artificial UV should be considered as a potential hazard for captive crocodilians if it is not properly managed, and this should be taken into account in the general design of facilities for reptilian husbandry.

Genetic, enzymatic and developmental alterations observed in Caiman latirostris exposed in ovo to pesticide formulations and mixtures in an experiment simulating environmental exposure.

In South America, economic interests in last years have produced a constant increase in transgenic soybean cropping, with the corresponding rise in pesticide formulated products. The aim of this study was to determine the effects of pesticides formulations and mixtures on a South American caiman, Caiman latirostris, after in ovo exposure. We conducted a field-like experiment which simulates the environmental exposure that a caiman nest can receive in neighbouring croplands habitats. Experimental groups were Control group, Treatment 1: sprayed with a glyphosate herbicide formulation, and Treatment 2: sprayed with a pesticide mixture of glyphosate, endosulfan and cypermethrin formulations. Results demonstrated genotoxicity, enzymatic and metabolic alterations, as well as growth delay in caimans exposed in ovo to Treatments 1 and 2, showing a higher toxicity for the mixture. Integral evaluation through biomarkers of different biological meaning is highly informative as early indicators of contamination with pesticides and mixtures in this wildlife species.

Embryological development of Caiman latirostris (Crocodylia: Alligatoridae).

A standard development embryological series is the primary basis to organize information of any embryological study and is also used to determine the age of eggs and embryos in field conditions. In this article, we calibrate developmental series of the broad-snouted caiman, Caiman latirostris, against an established series for Alligator mississippiensis. Morphometric measures and extend of the opaque-shell banding were also related to embryo age. In earlier stages, external morphological features alone can account for embryo age, but we suggest that morphometric measurements should be introduced later in the development. Unlike morphologic and morphometric attributes, the opaque patch was not a useful age predictor. As expected, a close correlation between embryonic development of C. latirostris and A. mississippiensis was observed.

Cranial sexual discrimination in hatchling broad-snouted caiman (Caiman latirostris)

Los neonatos de yacaré overo (Caiman latirostris) presentan un dimorfismo sexual en la forma y tamaño de su cráneo. Los neonatos machos tienen cráneos más pequeños que las hembras. Mediante el uso de análisis estadísticos multivariados es posible discriminar el sexo de los neonatos de yacaré overo en función de su forma y tamaño con una razonable eficiencia. La comprensión del dimorfismo sexual de los neonatos de crocodilianos podría ser mejorada mediante un abordaje experimental de variables genéticas y fenotípicas, tales como la temperatura de incubación y el nido de origen.

Broad-snouted caiman (Caiman latirostris) hatchlings present a consistent sexual dimorphism in their cranium shape and size. Male hatchlings have smaller crania than females. Using multivariate statistical analyses it is possible to discriminate sex in broad-snouted caiman hatchlings by their cranial shape with a reasonable efficiency. The understanding of sexual dimorphism of crocodilian hatchlings might be possibly improved by experimental approach considering, genetic and phenotypic variables such as incubation temperature and clutch of origin.

Cranial sexual discrimination in hatchling broad-snouted caiman (Caiman latirostris)

Los neonatos de yacaré overo (Caiman latirostris) presentan un dimorfismo sexual en la forma y tamaño de su cráneo. Los neonatos machos tienen cráneos más pequeños que las hembras. Mediante el uso de análisis estadísticos multivariados es posible discriminar el sexo de los neonatos de yacaré overo en función de su forma y tamaño con una razonable eficiencia. La comprensión del dimorfismo sexual de los neonatos de crocodilianos podría ser mejorada mediante un abordaje experimental de variables genéticas y fenotípicas, tales como la temperatura de incubación y el nido de origen.

Broad-snouted caiman (Caiman latirostris) hatchlings present a consistent sexual dimorphism in their cranium shape and size. Male hatchlings have smaller crania than females. Using multivariate statistical analyses it is possible to discriminate sex in broad-snouted caiman hatchlings by their cranial shape with a reasonable efficiency. The understanding of sexual dimorphism of crocodilian hatchlings might be possibly improved by experimental approach considering, genetic and phenotypic variables such as incubation temperature and clutch of origin.